Mus81-Mms4 (Mus81-Eme1 in some species) is a heterodimeric DNA structure-specific endonuclease that has been implicated in meiotic recombination and processing of damaged replication forks in fungi. We generated and characterized mutations in Drosophila melanogaster mus81 and mms4. Unlike the case in fungi, we did not find any role for MUS81-MMS4 in meiotic crossing over. A possible role for this endonuclease in repairing double-strand breaks that arise during DNA replication is suggested by the finding that mus81 and mms4 mutants are hypersensitive to camptothecin; however, these mutants are not hypersensitive to other agents that generate lesions that slow or block DNA replication. In fungi, mus81, mms4, and eme1 mutations are synthetically lethal with mutations in genes encoding RecQ helicase homologs. Similarly, we found that mutations in Drosophila mus81 and mms4 are synthetically lethal with null mutations in mus309, which encodes the ortholog of the Bloom Syndrome helicase. Synthetic lethality is associated with high levels of apoptosis in proliferating tissues. Lethality and elevated apoptosis were partially suppressed by a mutation in spn-A, which encodes the ortholog of the strand invasion protein Rad51. These findings provide insights into the causes of synthetic lethality.